This invention relates generally to fluorescent dye compounds useful as molecular probes. More specifically, this invention relates to 4,7-dichlororhodamine dyes useful as fluorescent labeling reagents.
ABI PRISM(trademark) 377 DNA Sequencer User""s Manual, Rev. A, Chapter 2, The Perkin-Elmer Corporation, Foster City, Calif. (p/n 903433) (1995).
Bergot, J. B., etal., U.S. Pat. No. 5,366,860 (1994)
Bergstrom, etal., JACS, 111: 374-375 (1989)
Caskey etal., U.S. Pat. No. 5,364,759 (1994)
Connell etal., Biotechniques, 5:342-348 (1987)
Eckstein ed., Oligonucleotides and Analogs, Chapters 8 and 9, IRL Press (1991)
Eckstein, Oligonucleotides and Analogues, IRL Press (1991)
Fung etal., U.S. Pat. No. 4,757,141 (1988)
Fung etal., U.S. Pat. No. 4,855,225 (1989)
Gait, Oligonucleotide Synthesis, IRL Press (1990)
Gebeyehu etal, Nucleic Acids Research, 15:4513-4535 (1987)
Gibson etal., Nucleic Acids Research, 15:6455-6467 (1987)
Haralambidis etal, Nucleic Acids Research, 15:4856-4876 (1987)
Haugland, Molecular Probes Handbook of Fluorescent Probes and Research Chemicals, Molecular Probes, Inc. (1992)
Herrmann, R., Josel, H., Drexhage, K., Arden, J., U.S. Pat. No. 5,750,409, issued May 12, 1998.
Hermanson, Bioconjugate Techniques, Academic Press (1996)
Hobbs etal., J. Org. Chem., 54:3420 (1989)
Hobbs etal., U.S. Pat. No. 5,151,507 (1992)
Hunkapiller, etal., U.S. Pat. No. 4,811,218(1989)
Innis etal. eds., PCR Protocols, Academic Press (1990)
Ju etal., Proc. Natl. Acad. Sci. USA 92:4347-4351 (1995)
Kasai, etal., Anal. Chem., 47:34037 (1975)
Khan, S., Menchen, S., Rosenbblum, B. xe2x80x9cSubstituted propargylethoxyamido nucleosides, oligonucleotides and methods for using samexe2x80x9d, U.S. Pat. No. 5,770,716, issued Jun. 23, 1998
Khan, S., Menchen, S., Rosenblum, B. xe2x80x9cPropargylethoxyamino nucleotidesxe2x80x9d, U.S. Pat. No. 5,821,356, issued Oct. 13, 1998
Khan, S. etal, xe2x80x9cNucleotides including a rigid linkerxe2x80x9d, Ser. No. 09,172,789, filing date Oct 14, 1998.
Khanna, etal., U.S. Pat. No. 4,318,846 (1988)
Lee etal. Nucleic Acids Research, 21:3761-3766 (1993)
Lee, L., Benson, S., Rosenblum, B., Spurgeon, S., Cassel, J. and Graham, R., xe2x80x9c4,7-Dichlororhodamine Dyesxe2x80x9d, U.S. Pat. No. 5,847,162, issued Dec. 8, 1998
Madabhushi, etal., International Patent Application No. WO US94/13852 (1994)
Maniatis, Methods in Enzymology, 65:299-305 (1980)
Menchen, etal., U.S. Pat. No. 5,188,934 (1993)
Mullis, U.S. Pat. No. 4,683,202 (1987)
Nelson etal., Nucleosides and Nucleotides, 5(3):233-241 (1986)
Nelson, etal., Nucleic Acids Research 20(23):6253-6259 (1992a)
Nelson, U.S. Pat. No. 5,141,813 (1992b)
Nelson,,U.S. Pat. No. 5,401,837 (1995)
Orgel etal., Nucleic Acids Research 11(18):6513 (1983)
Osterman, Methods of Protein and Nucleic Acid Research, Vol. 1 Springer-Verlag (1984)
Pringle etal., DNA Core Facilities Newsletter, 1:15-21 (1988)
Prober etal., Science, 238:336-341 (1987)
Rickwood and Hames, eds., Gel Electrophoresis of Nucleic Acids: A Practical Approach, IRL Press (1981)
Sanger, etal., Proc. Natl. Acad. Sci. USA 74:5463-5467 (1977)
Scheit, Nucleotide Analogs, John Wiley (1980)
Smith etal., Nucleic Acids Research, 113:2399-2412 (1985)
Smith etal., U.S. Pat. No. 5,118,800 (1992)
Steiner ed., Excited States of Biopolymers, Plenum Press (1983)
Stryer, Biochemistry, W. H. Freeman (1981)
Vos etal., Nucleic Acids Research, 23(21):4407-4414 (1995)
Vogel, M., Rettig, W., Sens, R, Drexhage, K., Chemical Physics Letters, 147:452-60 (1988)
Ward, etal., U.S. Pat. No. 5,559,767 (1995)
Webber, U.S. Pat. No. 5,075,217 (1991)
Wheeless etal, Flow Cytometry: Instrumentation and Data Analysis, pgs. 21-76, Academic Press (1985)
Woo, etal., U.S. Pat. No. 5,231,191 (1993)
The non-radioactive detection of biological analytes is an important technology in modern analytical biotechnology. By eliminating the need for radioactive labels, safety is enhanced and the environmental impact of reagent disposal is greatly reduced, resulting in decreased costs for analysis. Examples of methods utilizing such non-radioactive detection methods include DNA sequencing, oligonucleotide probe methods, detection of polymerase-chain-reaction products, immunoassays, and the like.
In many applications the independent detection of multiple spatially overlapping analytes in a mixture is required, e.g., single-tube multiplex DNA probe assays, immuno assays, multicolor DNA sequencing methods, and the like. In the case of multi-loci DNA probe assays, by providing multicolor detection, the number of reaction tubes may be reduced thereby simplifying the experimental protocols and facilitating the manufacturing of application-specific kits. In the case of automated DNA sequencing, multicolor labeling allows for the analysis of all four bases in a single lane thereby increasing throughput over single-color methods and eliminating uncertainties associated with inter-lane electrophoretic mobility variations.
Multiplex detection imposes a number of severe constraints on the selection of dye labels, particularly for analyses requiring an electrophoretic separation and treatment with enzymes, e.g., DNA sequencing. First, it is difficult to find a collection of dyes whose emission spectra are spectrally resolved, since the typical emission band half-width for organic fluorescent dyes is about 40-80 nanometers (nm) and the width of the available spectrum is limited by the excitation light source. Second, even if dyes with non-overlapping emission spectra are found, the set may still not be suitable if the respective fluorescent efficiencies are too low. For example, in the case of DNA sequencing, increased sample loading cannot compensate for low fluorescence efficiencies (Pringle). Third, when several fluorescent dyes are used concurrently, simultaneous excitation becomes difficult because the absorption bands of the dyes are widely separated. Fourth, the charge, molecular size, and conformation of the dyes must not adversely affect the electrophoretic mobilities of the fragments. Finally, the fluorescent dyes must be compatible with the chemistry used to create or manipulate the fragments, e.g., DNA synthesis solvents and reagents, buffers, polymerase enzymes, ligase enzymes, and the like.
Because of these severe constraints only a few sets of fluorescent dyes have been found that can be used in multicolor applications, particularly in the area of four-color DNA sequencing (Smith 1992, 1995; Prober; Connell).
One class of fluorescent dyes particularly useful in multicolor applications are the rhodamine dyes, e.g., tetratmethylrhodamine (TAMRA), rhodamine X (ROX), rhodamine 6G (R6G), rhodamine 110 (R110), and the like (Bergot). Rhodamine dyes are particularly attractive relative to fluorescein dyes because (1) rhodamines are typically more photostable than fluoresceins, (2) rhodamine-labeled dideoxynucleotides are better substrates for thermostable polymerase enzymes, and (3) the emission spectra of rhodamine dyes is significantly to the red (higher wavelength) of fluoresceins.
However, one important drawback of presently available rhodamine dyes in the context of multiplex detection methods is the relatively broad emission spectrum of such dyes. This broad emission spectrum results in poor spectral resolution between spectrally neighboring dyes thereby making the multicomponent analysis of such dye combinations difficult. The fluorescence emission spectra shown in FIG. 7A demonstrate this high degree of spectral overlap. A second drawback of currently available rhodamine dyes is that their absorption spectrum does not match the wavelength of currently available solid state frequency-doubled green diode lasers, e.g., neodymium solid-state YAG lasers, which have an emission line at approximately 532 nm. It is highly advantageous to use such lasers because of their compact size, long useful life, and efficient use of power.
The present invention is directed towards our discovery of a class of 4,7-dichlororhodamine dyes useful as molecular probes.
It is an object of the invention to provide a class of rhodamine dyes which have emission spectra which are substantially narrower than presently available rhodamine dyes.
It is another object of the invention to provide a class of rhodamine dyes which have an absorption spectrum shifted to the red as compared to existing rhodamine dyes.
In a first aspect, the foregoing and other objects of the invention are achieved by a compound having the formula: 
In a second aspect, the invention includes a compound having the formula: 
In a third aspect, the invention includes a labeled nucleotide having the formula: 
In a fourth aspect, the invention includes a labeled polynucleotide containing a nucleotide having the formula: 
The linkage linking B and D is attached to D at one of positions R1-R6 or X1-X3. Preferably, the linkage linking B and D is attached to D at one of positions X2 or X3. In a particularly preferred embodiment, the linkage is 
If B is a purine, the linkage is attached to the 8-position of the purine. If B is 7-deazapurine, the linkage is attached to the 7-position of the 7-deazapurine. If B is pyrimidine, the linkage is attached to the 5-position of the pyrimidine.
In a fifth aspect, the present invention includes a method of polynucleotide sequencing, such method including the following steps. Forming a mixture of a first, a second, a third, and a fourth class of polynucleotides such that each polynucleotide in the first class includes a 3xe2x80x2-terminal dideoxyadenosine and is labeled with a first dye, each polynucleotide in the second class includes a 3xe2x80x2-terminal dideoxycytidine and is labeled with a second dye, each polynucleotide in the third class includes a 3xe2x80x2-terminal dideoxyguanosine and is labeled with a third dye, and, each polynucleotide in the fourth class includes a 3xe2x80x2-terminal dideoxythymidine and is labeled with a fourth dye. The dyes are selected such that one of the first, second, third, or fourth dyes is a 4,7-dichlororhodamine dye of the invention, the other of the dyes being spectrally resolvable from the 4,7-dichlororhodamine dye and from each other. Electrophoretically separating the polynucleotides thereby forming bands of similarly sized polynucleotides, illuminating the bands with an illumination beam capable of causing the dyes to fluoresce, and, identifying the classes of the polynucleotides in the bands by the fluorescence spectrum of the dyes.
These and other aspects, objects, features, and advantages of the present invention will become better understood with reference to the following description, drawings, and appended claims.
Reference will now be made in detail to the preferred embodiments of the invention, examples of which are illustrated in the accompanying drawings. While the invention will be described in conjunction with the preferred embodiments, it will be understood that they are not intended to limit the invention to those embodiments. On the contrary, the invention is intended to cover alternatives, modifications, and equivalents, which may be included within the invention as defined by the appended claims.
Generally, the present invention comprises a novel class of 4,7-dichlororhodamine compounds useful as fluorescent dyes, reagents employing such dyes as molecular labels, and methods utilizing such dyes and reagents in the area of analytical biotechnology. The compounds of the present invention find particular application in the area of multicolor fluorescent DNA sequencing and fragment analysis.
The invention is based in part on the discovery that the fluorescent properties of 4,7-dichlororhodamines and related dyes are highly favorable for use as molecular probes. Their emission band widths are generally 20-30 percent narrower than analogs lacking the 4,7-dichloro derivatives, and, their emission and absorption maxima are at wavelengths generally about 10-30 nm higher than analogs lacking the 4,7-dichloro derivatives.
I. Definitions
Unless stated otherwise, the following terms and phrases as used herein are intended to have the following meanings:
xe2x80x9cLinking groupxe2x80x9d (L) refers to a functionality capable of reacting with a xe2x80x9ccomplementary functionalityxe2x80x9d attached to a reagent, such reaction forming a xe2x80x9clinkagexe2x80x9d connecting a dye to a reagent. The particular linking group used depends on the nature of the complementary functionality and the type of linkage desired. In some cases, the linking group must be activated prior to reaction with a complementary functionality, e.g., the activation of a carboxylate linking group with dicyclohexylcarbodiimide and N-hydroxysuccinimide to form a N-hydroxysuccinimide (NHS) ester. Preferably, whenever the complementary functionality is amine, the linking group of the invention is isothiocyanate, isocyanate, acyl azide, NHS ester, sulfonyl chloride, aldehyde or glyoxal, epoxide, carbonate, aryl halide, imidoester, carbodiimide, anhydride, 4,6-dichlorotriazinylamine, or other active carboxylate. Preferably, whenever the complementary functionality is sulfhydryl, the linking group is haloacetyl, alkyl halide, maleimide, halo acetyl, aziridine, acryloyl, arylating agent, e.g., fluorobenzene, and the like. When the complementary functionality is carboxylate, the linking group is preferably diazoalane, diazoacetyl, carbonyldiimidazole, and carbodiimide (Hermanson). In a particularly preferred embodiment, the linking group is an activated NHS ester which reacts with an amine complementary functionality, where to form the activated NHS ester, a dye of the invention including a carboxylate linking group is reacted with dicyclohexylcarbodiimide and N-hydroxysuccinimide to form the NHS ester (Khanna; Kasai). Table 1 below shows a sampling of representative linking groups along with compatible complementary functionalities and resulting linkages.
The term xe2x80x9clower alkylxe2x80x9d denotes straight-chain and branched hydrocarbon moieties containing from 1 to 8 carbon atoms, i.e., methyl, ethyl, propyl, isopropyl, tert-butyl, isobutyl, sec-butyl, neopentyl, tert-pentyl, and the like.
The term xe2x80x9cpropanoxe2x80x9d in particular refers to the moiety xe2x80x94CH2CH2CH2xe2x80x94.
xe2x80x9cCycloalkylxe2x80x9d refers to hydrocarbon moieties that form rings, e.g. cyclohexyl and cyclopentyl. Nitrogen atoms with cycloalkyl substituents may form aziridinyl, azetidinyl, pyrrolidinyl, piperidinyl, larger rings, and substituted forms thereof
xe2x80x9cLower substituted alkylxe2x80x9d denotes a lower alkyl including electron-withdrawing substituents, such as halo, cyano, nitro, sulfo, and the like.
xe2x80x9cLower haloalkylxe2x80x9d denotes a lower substituted alkyl with one or more halogen atom substituents, usually fluoro, chloro, bromo, or iodo.
xe2x80x9cLower alkenexe2x80x9d denotes a lower alkyl or lower substituted alkyl wherein one or more of the carbon-carbon bonds is a double bond.
xe2x80x9cLower alkynexe2x80x9d denotes a lower alkyl or lower substituted alkyl wherein one or more of the carbon-carbon bonds is a triple bond.
xe2x80x9cLower Alkoxyxe2x80x9d refers to a moiety including lower alkyl single bonded to an oxygen atom.
xe2x80x9cArylxe2x80x9d refers to single or multiple phenyl or substituted phenyl, e.g., benzene, naphthalene, anthracene, biphenyl, and the like.
The term xe2x80x9cnucleosidexe2x80x9d refers to a compound consisting of a purine, deazapurine, or pyrimidine nucleoside base, e.g., adenine, guanine, cytosine, uracil, thymine, deazaadenine, deazaguanosine, and the like, linked to a pentose at the 1xe2x80x2 position, including 2xe2x80x2-deoxy and 2xe2x80x2-hydroxyl forms (Stryer). The term xe2x80x9cnucleotidexe2x80x9d as used herein refers to a phosphate ester of a nucleoside, e.g., triphosphate esters, wherein the most common site of esterification is the hydroxyl group attached at the C-5 position of the pentose. Many times in the present disclosure the term nucleoside will be intended to include both nucleosides and nucleotides.
xe2x80x9cAnalogsxe2x80x9d in reference to nucleosides include synthetic analogs having modified base moieties, modified sugar moieties, and/or modified phosphate ester moieties, e.g., as described elsewhere (Scheit; Eckstein). The term xe2x80x9clabeled nucleosidexe2x80x9d refers to nucleosides which are covalently attached to the dye compounds of Formula I.
As used herein, the terms xe2x80x9cpolynucleotidexe2x80x9d or xe2x80x9coligonucleotidexe2x80x9d refer to linear polymers of natural nucleotide monomers or analogs thereof including double and single stranded deoxyribonucleotides, ribonucleotides, a-anomeric forms thereof and the like. Usually the nucleoside monomers are linked by phosphodiester linkages, where as used herein, the term xe2x80x9cphosphodiester linkagexe2x80x9d refers to phosphodiester bonds or analogs thereof including phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate, phosphoranilidate, phosphoramidate, and the like, including associated counterions, e.g., H+, NH4+, Na+, and the like if such counterions are present. Polynucleotides typically range in size from a few monomeric units, e.g. 8-40, to several thousands of monomeric units. Whenever a polynucleotide is represented by a sequence of letters, such as xe2x80x9cATGCCTG,xe2x80x9d it will be understood that the nucleotides are in 5xe2x80x2xe2x86x923xe2x80x2 order from left to right and that xe2x80x9cAxe2x80x9d denotes deoxyadenosine, xe2x80x9cCxe2x80x9d denotes deoxycytidine, xe2x80x9cGxe2x80x9d denotes deoxyguanosine, and xe2x80x9cTxe2x80x9d denotes thymidine, unless otherwise noted.
As used herein the term xe2x80x9cspectral resolutionxe2x80x9d in reference to a set of dyes means that the fluorescent emission spectra of the dyes are sufficiently distinct, i.e., sufficiently non-overlapping, that reagents to which the respective dyes are attached, e.g., polynucleotides, can be distinguished on the basis of the fluorescent signal generated by the respective dyes using standard photodetection systems, e.g., employing a system of band pass filters and photomultiplier tubes, a charged-coupled device in conjunction with a spectrograph, or the like, as exemplified by systems described elsewhere (Hunkapiller; Wheeless).
The term xe2x80x9csubstitutedxe2x80x9d as used herein refers to a molecule wherein one or more hydrogen atoms are replaced with one or more non-hydrogen atoms, functional groups or moieties. For example, an unsubstituted nitrogen is xe2x80x94NH2, while a substituted nitrogen is xe2x80x94NHCH3. Exemplary substituents include but are not limited to halo, e.g., fluorine and chlorine, lower alkyl, lower alkene, lower alkyne, sulfate, sulfonate, sulfone, amino, ammonium, amido, nitrile, lower alkoxy, phenoxy, aromatic, phenyl, polycyclic aromatic, heterocycle, and linking group.
II. 4,7-Dichlororhodamine Dye Compounds
In a first aspect, the present invention comprises a novel class of 4,7-dichlororhodamine dye compounds having the general structure shown immediately below as Formula I. (Note that all molecular structures provided throughout this disclosure are intended to encompass not only the exact electronic structure presented, but also include all resonant structures, enantiomers, diastereomers, and protonation states thereof.) 
In Formula I, the variable substituents are defined as follows. R1-R6 taken separately are hydrogen, fluorine, chlorine, lower alkyl, lower alkene, lower alkyne, cycloalkyl, phenyl, aryl, sulfonate, sulfone, amino, amido, nitrile, lower alkoxy, linking group, or combinations thereof or, when taken together, R1 and R6 is benzo, or, when taken together, R4 and R5 is benzo. Preferably, R1-R6 are hydrogen, methyl, or ethyl. Y1-Y4 taken separately are hydrogen, lower alkyl, alkyl sulfonate, alkyl carboxylate, or cycloalkyl. Or, when taken together, Y1 and R2, Y2 and R1, Y3 and R3, and/or Y4 and R4 are propano, ethano, or substituted forms thereof to form fused rings. X1-X3 taken separately are hydrogen, chlorine, fluorine, lower alkyl, carboxylate, sulfonic acid (sulfonate), hydroxymethyl (xe2x80x94CH2OH), and linking groups. Preferably, X1 is carboxylate and X2 and X3 taken separately are hydrogen and linking group.
In a set of particularly preferred compound of the present invention, R is hydrogen (dJON) or methyl (DMDJ), shown below as FORMULA II. 
Another particularly preferred compound of the present invention is referred to as dR650, shown below as FORMULA III. 
A third particularly preferred set of compounds are where R is 6-hexanoic acid (dJODA) or methyl-p-benzoic acid (dR134), as shown below in FORMULA IV. 
A fourth particularly preferred compound of the present invention is referred to herein as dR139, where Y1 and Y2, and Y3 and Y4, form pyrrolidinyl rings as nitrogen substituents, R1-6 are hydrogen, X1 is carboxyl, and X2 and X3 are carboxyl and hydrogen. The structure of dR139 is shown below as Formula V. 
In a second aspect, the present invention comprises a novel class of 4,7-dichlororhodamine dye compounds having the general structure shown below as FORMULA VI. 
In Formula VI, R7-R10, R12-R16, and R18 may be hydrogen, fluorine, chlorine, methyl, ethyl, lower alkyl, lower alkene, lower alkyne, cycloalkyl, phenyl, aryl, sulfonate, sulfone, amino, amido, nitrile, lower alkoxy, liking group, or combinations thereof Preferably, R7-R10 and R13-R16 are hydrogen, methyl, or ethyl. R7 and R8, or R13 and R14, taken together may be oxygen (xe2x95x90O), sulfur (xe2x95x90S), imminium (xe2x95x90NH), alkylimminium (xe2x95x90NR). R11 and R17 may be hydrogen, lower alkyl, alkyl sulfonate, alkyl carboxylate, lower alkene, lower alkyne, cycloalkyl, phenyl, aryl, linking group, or combinations thereof Preferably R11 and R17 are methyl or phenyl. X1-X3 taken separately are hydrogen, chlorine, fluorine, lower alkyl, amine, amide, carboxylate, sulfonic acid (sulfonate), hydroxymethyl (xe2x80x94CH2OH), and linking groups. Preferably, X1 is carboxylate and X2 and X3 taken separately are hydrogen or linking group. Particular preferred embodiments are where R7, R8, R10 R13, R14, and R17 are hydrogen or methyl, R9 and R15 are hydrogen, R11 and R15 are methyl or phenyl, and R12 and R18 are hydrogen.
III. Reagents Utilizing 4,7-Dichlororhodamine Dye Compounds
In another aspect, the present invention comprises reagents labeled with the 4,7-dichlororhodamine dye compounds of Formulas I-VI. Reagents of the invention can be virtually anything to which the dyes of the invention can be attached. Preferably the dyes are covalently attached to the reagent directly or through a linkage. Exemplary reagents include proteins, polypeptides, polysaccharides, nucleotides, nucleosides, polynucleotides, lipids, solid supports, organic and inorganic polymers, and combinations and assemblages thereof such as chromosomes, nuclei, living cells, such as bacteria, other microorganisms, mammalian cells, tissues, glycoproteins, and the like.
A. Nucleotide Reagents
A preferred class of reagents of the present invention comprise nucleotides and nucleosides which incorporate the asymmetric benzoxanthene dyes of the invention. Such nucleotide/side reagents are particularly useful in the context of labeling polynucleotides formed by enzymatic synthesis, e.g., nucleotide triphosphates used in the context of PCR amplification, Sanger-type polynucleotide sequencing, and nick-translation reactions.
Preferred nucleotide/side reagents of the present invention are shown below in Formula VII wherein 
B is a nucleoside base; 7-deazapurine, purine, or pyrimidine nucleotide base, analogs thereof, and preferably uracil, cytosine, deazaadenine, or deazaguanosine. D is the 4,7-dichlororhodamine dye compound of the Formulas I-VI of the invention. W1 and W2 taken separately are H or OH. W3 is OH, OPO3, OP2O6, OP3O9, including analogs thereof, e.g., phosphorothioate, phosphoroanilidate, phosphoroanilothioate, phosphoramidiate, and other like phosphate analogs, including associated counterions if present, e.g., H+, Na+, NH4+, and the like. In one preferred embodiment, W1 is H, W2 is OH, and W3 is OP3O9. In a second preferred embodiment, W1 and W2 are H and W3 is OP3O9. When B is purine or 7-deazapurine, the sugar moiety is attached at the N9-position of the purine or deazapurine, and when B is pyrimidine, the sugar moiety is attached at the N1-position of the pyrimidine. The linkage linking B and D is attached to D at one of positions R1-R18 or X1-X3.
When B is purine or 7-deazapurine, the sugar moiety is attached at the N9-position of the purine or deazapurine, and when B is pyrmidine, the sugar moiety is attached at the N1-position of the pyrimidine.
The linkage linking B and D may be attached to D at any one of positions R1-R18 or X1-X3. Preferably, the linkage is attached at one of X2 or X3. Preferably, when B is a purine, the linkage linking B and D is attached to the 8-position of the purine, when B is 7-deazapurine, the linkage is attached to the 7-position of the 7-deazapurine, and when B is pyrimidine, the linkage is attached to the 5-position of the pyrmidine.
In one particularly preferred embodiment, the nucleotides of the present invention are dideoxynucleotide triphosphates having the structure shown below in Formula VIII, including associated counterions if present. 
Labeled dideoxy nucleotides such as that shown in Formula VIII find particular application as chain terminating agents, or xe2x80x9cterminatorsxe2x80x9d, in Sanger-type DNA sequencing methods (Sanger).
In a second particularly preferred embodiment, the nucleotides of the present invention are deoxynucleotide triphosphates having the structure shown in Formula IX below, including associated counterions if present. 
Labeled deoxynucleotides such as that shown in Formula IX find particular application as means for labeling polymerase extension products, e.g., in the polymerase chain reaction (Mullis).
Nucleotide/side labeling can be accomplished using any of a large number of known nucleoside/tide labeling techniques using known linking groups, and associated complementary functionalities to form linkages. See above for a discussion of preferred linking groups. The linkage linking the dye and nucleoside should (i) not interfere with oligonucleotide-target hybridization, (ii) be compatible with relevant enzymes, e.g., polymerases, ligases, and the like, and (iii) not quench the fluorescence of the dye.
In one preferred embodiment, the dyes of the invention are covalently linked to the 5-carbon of pyrimidine bases or to the 7-carbon of 7-deazapurine bases. Several suitable base labeling procedures have been reported that can be used with the invention. (Gibson; Gebeyehu; Haralambidis; Nelson 1992; Bergstrom; Fung 1988; Ward; Woo.)
Preferably, the linkages are acetylenic amido or alkenic amido linkages, the linkage between the dye and the nucleotide base being formed by reacting an activated NHS ester of the dye with an alkynylamino- or alkenylamino-derivatized base of a nucleotide. More preferably, the resulting linkage is 3carboxy)amino-1-propynyl or 3-amino-1-propyn-1-yl (Formula X.1). Several preferred linkages for linking the dyes of the invention to a nucleoside base are shown below as Formulas X.1-6 (Khan). 
The synthesis of alkynylamino-derivatized nucleosides is described by (Hobbs 1989, 1992). Briefly, the alkynylamino-derivatized nucleotides are formed by placing the appropriate halodideoxynucleoside (usually 5-iodopyrimidine and 7-iodo-7-deazapurine dideoxynucleosides) and Cu(I) in a flask, flushing with argon to remove air, adding dry DMF, followed by addition of an alkynylamine, triethylamine and Pdxc2x0. The reaction mixture is stirred for several hours, or until thin layer chromatography indicates consumption of the halodideoxynucleoside. When an unprotected alkynylamine is used, the alkynylamino-nucleoside can be isolated by concentrating the reaction mixture and chromatographing on silica gel using an eluting solvent which contains ammonium hydroxide to neutralize the hydrohalide generated in the coupling reaction. When a protected alkynylamine is used, methanol/methylene chloride can be added to the reaction mixture, followed by the bicarbonate form of a strongly basic anion exchange resin. The slurry can then be stirred for about 45 minutes, filtered, and the resin rinsed with additional methanol/methylene chloride. The combined filtrates can be concentrated and purified by flash-chromatography on silica gel using a methanol-methylene chloride gradient. The 5xe2x80x2-triphosphates are obtained by standard techniques.
B. Polynucleotide Reagents
Yet another preferred class of reagents of the present invention comprise polynucleotides labeled with the 4,7-dichlororhodamine dyes of the invention. Such labeled polynucleotides are useful in a number of important contexts including as DNA sequencing primers, PCR primers, oligonucleotide hybridization probes, and the like.
The polynucleotides of the invention include a nucleotide having the formula: 
wherein the variable substituents and linkages are defined as follows. D is a 4,7-dichlororhodamine dye compound of the invention. B is a 7-deazapurine, purine, or pyrimidine nucleotide base, preferably uracil, cytosine, deazaadenine, or deazaguanosine. Z1 is H, OH, or OCH3. Z2 is H, OH, OPO3, OP2O6, OP3O9, or Nuc, a neighboring nucleotide, wherein Nuc and the nucleoside are linked by a phosphodiester linkage or analog thereof, e.g., phosphorothioate, phosphoroanilidate, phosphoroanilothioate, phosphoramidiate, and other like phosphate analogs, including associated counterions if present, e.g., H+, Na+, NH4+, the linkage being attached to the 5xe2x80x2-position of Nuc. Z3 is H, OPO2, including phosphate analogs, or Nuc, wherein Nuc and the nucleoside are linked by a phosphodiester linkage or analog thereof, the linkage being attached to the 3xe2x80x2-position of Nuc wherein Nuc refers to a nucleoside, nucleotide, or polynucleotide. When B is purine or 7-deazapurine, the sugar moiety is attached at the N9-position of the purine or deazapurine, and when B is pyrimidine, the sugar moiety is attached at the N1-position of the pyrimidine. B is attached to the sugar moiety and to the dye compound as described above for the nucleotide reagent of the invention. As defined, the labeled nucleotide of Formula XI can be the 5xe2x80x2-terminal nucleotide, the 3xe2x80x2-terminal nucleotide, or any internal nucleotide of the polynucleotide.
In one preferred embodiment, the polynucleotide of the present invention includes multiple dyes, at least one of which is a dye compound of the invention, located such that fluorescence energy transfer takes place between a donor dye and an acceptor dye. Such multi-dye polynucleotides find application as spectrally-tunable probes or DNA sequencing primers (Ju; Lee).
Labeled polynucleotides may be synthesized either enzymatically, e.g., using a DNA polymerase or ligase (Stryer), or by chemical synthesis, e.g., by the phosphoramidite method, the phosphite-triester method, and the like (Gait). Labels may be introduced during enzymatic synthesis utilizing labeled nucleotide triphosphate monomers as described above or may be introduced subsequent to synthesis.
Generally, if the labeled polynucleotide is made by enzymatic synthesis, the following procedure may be used. A template DNA is denatured and an oligonucleotide primer is annealed to the template DNA. A mixture of deoxynucleotide triphosphates and/or dideoxynucleotide triphosphates is added to the reaction including dGTP, dATP, dCTP, ddTTP, ddGTP, ddATP, ddCTP, and ddTTP, where at least a fraction of one of at least one the deoxynucleotides and/or dideoxynucleotides is labeled with a dye compound of the invention as described above. Next, a polymerase enzyme is added under conditions where its polymerase activity is operative. A labeled polynucleotide is formed by the incorporation of the labeled deoxynucleotides and/or dideoxynucleotides during polymerase strand synthesis. In an alternative enzymatic synthesis method, two primers are used instead of one, one primer complementary to the + (plus) strand and the other complementary to the xe2x88x92 (minus) strand of the target, the polymerase is a thermostable polymerase, and the reaction temperature is cycled between a denaturation temperature and an extension temperature, thereby exponentially synthesizing a labeled complement to the target sequence by PCR (Mullis; Innis).
Subsequent to synthesis, the polynucleotide may be labeled at a number of positions including the 5xe2x80x2-terminus (Eckstein; Orgel; Smith); the phosphodiester backbone (Eckstein); or at the 3xe2x80x2-terminus (Nelson 1992a; Nelson 1992b; Nelson 1995). For a through review of oligonucleotide labeling procedures see (Steiner).
In one preferred post-synthesis chemical labeling method an oligonucleotide is labeled as follows. A dye including a carboxylate linking group is converted to the NHS ester by reacting with approximately 1 equivalent of 1,3-dicyclohexylcarbodiimide and approximately 3 equivalents of N-hydroxysuccinimide in dry ethyl acetate for 3 hours at room temperature. The reaction mixture is washed with 5% HCl, dried over magnesium sulfate, filtered, and concentrated to a solid which is resuspended in DMSO. The DMSO dye stock is then added in excess (10-20xc3x97) to an aminohexyl derivatized oligonucleotide in 0.25 M bicarbonate/carbonate buffer at pH 9.4 and allowed to react for 6 hours (Fung 1988). The dye labeled oligonucleotide is separated from unreacted dye by passage through a size-exclusion chromatography column eluting with buffer, e.g., 0.1 molar triethylammonium acetate (TEAA). The fraction containing the crude labeled oligonucleotide is further purified by reverse phase HPLC employing gradient elution.
IV. Methods Utilizing Compounds and Reagents of the Invention
The dyes and reagents of the present invention are well suited to methods utilizing fluorescent detection, particularly methods requiring the simultaneous detection of multiple spatially-overlapping analytes. Dyes and reagents of the invention are particularly well suited for identifying classes of polynucleotides that have been subjected to a biochemical separation procedure, such as electrophoresis, where a series of bands or spots of target substances having similar physiochemical properties, e.g. size, conformation, charge, hydrophobicity, or the like, are present in a linear or planar arrangement. As used herein, the term xe2x80x9cbandsxe2x80x9d includes any spatial grouping or aggregation of analytes on the basis of similar or identical physiochemical properties. Usually bands arise in the separation of dye-polynucleotide conjugates by electrophoresis.
Classes of polynucleotides can arise in a variety of contexts. In a preferred category of methods referred to herein as xe2x80x9cfragment analysisxe2x80x9d or xe2x80x9cgenetic analysisxe2x80x9d methods, labeled polynucleotide fragments are generated through template-directed enzymatic synthesis using labeled primers or nucleotides, e.g., by ligation or polymerase-directed primer extension; the fragments are subjected to a size-dependent separation process, e.g., electrophoresis or chromatography; and, the separated fragments are detected subsequent to the separation, e.g., by laser-induced fluorescence. In a particularly preferred embodiment, multiple classes of polynucleotides are separated simultaneously and the different classes are distinguished by spectrally resolvable labels.
One such fragment analysis method known as amplified fragment length polymorphism detection (AmpFLP) is based on amplified fragment length polymorphisms, i.e., restriction fragment length polymorphisms that are amplified by PCR (Vos). These amplified fragments of varying size serve as linked markers for following mutant genes through families. The closer the amplified fragment is to the mutant gene on the chromosome, the higher the linkage correlation. Because genes for many genetic disorders have not been identified, these linkage markers serve to help evaluate disease risk or paternity. In the AmpFLPs technique, the polynucleotides may be labeled by using a labeled polynucleotide PCR primer, or by utilizing labeled nucleotide triphosphates in the PCR.
Another exemplary fragment analysis method is based on variable number of tandem repeats; or VNTRs (Webber; Caskey). VNTRs are regions of double-stranded DNA that contain adjacent multiple copies of a particular sequence, with the number of repeating units being variable. Examples of VNTR loci are pYNZ22, pMCT118, and Apo B. A subset of VNTR methods are those methods based on the detection of microsatellite repeats, or short tandem repeats (STRs), i.e., tandem repeats of DNA characterized by a short (2-4 bases) repeated sequence. One of the most abundant interspersed repetitive DNA families in humans is the (dC-dA)nxe2x80x94(dG-dT)n dinucleotide repeat family (also called the (CA)n dinucleotide repeat family). There are thought to be as many as 50,000 to 100,000 (CA)n repeat regions in the human genome, typically with 15-30 repeats per block. Many of these repeat regions are polymorphic in length and can therefore serve as useful genetic markers. Preferably, in VNTR or STR methods, a dye label is introduced into the polynucleotide fragments by using a dye-labeled PCR primer.
In a particularly preferred fragment analysis method, classes identified in accordance with the invention are defined in terms of terminal nucleotides so that a correspondence is established between the four possible terminal bases and the members of a set of spectrally resolvable dyes (Fung 1989). Such sets are readily assembled from the dyes of the invention by measuring emission and absorption bandwidths with commercially available spectrophotometers. More preferably, the classes arise in the context of the chemical or chain termination methods of DNA sequencing, and most preferably the classes arise in the context of the chain termination method, i.e., dideoxy DNA sequencing, or Sanger sequencing. This method involves the synthesis of a DNA strand by a DNA polymerase in vitro using a single-stranded or double-stranded DNA template whose sequence is to be determined. Synthesis is initiated at only the one site where an oligonucleotide primer anneals to the template. The synthesis reaction is terminated by incorporation of a nucleotide analog that will not support continued DNA elongation. The chain-terminating nucleotide analogs are typically 2xe2x80x2,3xe2x80x2-dideoxynucleoside 5xe2x80x2-triphosphates (ddNTPs) which lack the 3xe2x80x2-OH group necessary for 3xe2x80x2 to 5xe2x80x2 DNA chain elongation. When proper proportions of dNTPs (2xe2x80x2-deoxynucleoside 5xe2x80x2-triphosphates),and one of the four ddNTPs are used, enzyme-catalyzed polymerization will be terminated in a fraction of the population of chains at each site where the ddNTP can be incorporated. If labeled primers or labeled ddNTPs are used for each reaction, the sequence information can be detected by fluorescence after separation by high-resolution electrophoresis. In the chain termination method, dyes of the invention can be attached to either sequencing primers or dideoxynucleotides.
In each of the above fragment analysis methods labeled polynucleotides are preferably separated by electrophoretic procedures (Rickwood and Hames; Osterman) Preferably the type of electrophoretic matrix is crosslinked or uncrosslinked polyacrylamide having a concentration (weight to volume) of between about 2-20 weight percent. More preferably, the polyacrylamide concentration is between about 4-8 percent. Preferably in the context of DNA sequencing in particular, the electrophoresis matrix includes a strand separating, or denaturing, agent, e.g., urea, formamide, and the like. Detailed procedures for constructing such matrices are given by (Maniatis 1980; ABI PRISM(trademark) 377 DNA Sequencer User""s Manual). The optimal polymer concentration, pH, temperature, concentration of denaturing agent, etc. employed in a particular separation depends on many factors, including the size range of the nucleic acids to be separated, their base compositions, whether they are single stranded or double stranded, and the nature of the classes for which information is sought by electrophoresis. Accordingly application of the invention may require standard preliminary testing to optimize conditions for particular separations.
Subsequent to electrophoretic separation, the dye-polynucleotide conjugates are detected by measuring the fluorescence emission from the dye labeled polynucleotides. To perform such detection, the labeled polynucleotides are illuminated by standard means, e.g. high intensity mercury vapor lamps, lasers, or the like. Preferably the illumination means is a laser having an illumination beam at a wavelength between 488 and 550 nm. More preferably, the dye-polynucleotides are illuminated by laser light generated by an argon ion laser, particularly the 488 and 514 nm emission lines of an argon ion laser, or the 532 emission line of a neodymium solid-state YAG laser. Several argon ion lasers are available commercially which lase simultaneously at these lines, e.g., the Model 2001 from Cyonics, Ltd. (Sunnyvale, Calif). The fluorescence is then detected by a light-sensitive detector, e.g., a photomultiplier tube, a charged coupled device, or the like.